SUMMARY Bacteria of the genus Rickettsia are transmitted from arthropod vectors and primarily infect cells of the mammalian endothelial system. Throughout this infectious cycle, the bacteria are exposed to the deleterious effects of serum complement. Using Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), as a model rickettsial species, we have previously demonstrated that this class of pathogen interacts with human factor H to mediate partial survival in human serum. Herein, we demonstrate that R. conorii also interacts with the terminal complement complex inhibitor vitronectin (Vn). We further demonstrate that an evolutionarily conserved rickettsial antigen, Adr1/RC1281, interacts with human vitronectin and is sufficient to mediate resistance to serum killing when expressed at the outer-membrane of serum sensitive E. coli. Adr1 is an integral outer-membrane protein whose structure is predicted to contain eight membrane-embedded β-strands and four “loop” regions that are exposed to extracellular milieu. Site-directed mutagenesis of Adr1 revealed that at least two predicted “loop” regions are required to mediate resistance to complement-mediated killing and vitronectin acquisition. These results demonstrate that rickettsial species have evolved multiple mechanisms to evade complement deposition and that evasion of killing in serum is an evolutionarily conserved virulence attribute for this genus of obligate intracellular pathogens.

The third meeting of WHO's Product Development for Vaccines Advisory Committee (PDVAC) was held in June 2016, with a remit to revisit the pathogen areas for which significant progress has occurred since recommendations from the 2015 meeting, as well as to consider new advances in the development of vaccines against other pathogens. Since the previous meeting, significant progress has been made with regulatory approvals of the first malaria and dengue vaccines, and the first phase III trials of a respiratory syncytial virus (RSV) vaccine candidate has started in the elderly and pregnant women. In addition, PDVAC has also supported vaccine development efforts against important emerging pathogens, including Middle Eastern Coronavirus (MERS CoV) and Zika virus. Trials of HIV and tuberculosis vaccine candidates are steadily progressing towards pivotal data points, and the leading norovirus vaccine candidate has entered a phase IIb efficacy study. WHO's Immunization, Vaccine and Biologicals (IVB) department is actively working in several pathogen areas on the recommendation of PDVAC, as well as continuing horizon scanning for advances in the development of vaccines that may benefit low and middle income countries (LMICs), such as the recent licensure of the enterovirus 71 (EV71) vaccine in China. Following on from discussions with WHO's Strategic Advisory Group of Experts (SAGE) on Immunization, PDVAC will also look beyond licensure and consider data needs for vaccine recommendation and implementation to reduce the delay between vaccine approval and vaccine impact.

Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium . In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium . However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15–30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.